Multivalent glycan scaffolds and methods of use thereof

Abstract

Multivalent glycan scaffolds, and methods of making, and compositions and methods of use thereof are provided. The multivalent glycan scaffolds are typically composed of an underlying branched scaffold such as a dendrimer, with two or more glycans and optionally one or more functional elements linked to the terminal ends thereof. The glycans can be synthetic or isolated from a natural source. Methods of using glycan scaffolds to identify binding partners such as cells and/or glycan binding proteins are also provided. A method of identifying glycan-based binding interactions can include contacting a target material with a known multivalent glycan scaffold and identifying element(s) from the target material that binds thereto. In other embodiments, a method of identifying glycan-based binding interactions includes contacting a known target material element with a library of different species of multivalent glycan scaffolds each having two or more glycans, and identifying glycan scaffold(s) that bind thereto.

We claim: 
     
         1 . A method of identifying glycan-based binding interactions comprising contacting a target material with a known multivalent glycan scaffold comprising two or more glycans, and identifying element(s) from the target material that binds thereto. 
     
     
         2 . The method of  claim 1 , wherein the target material is a population of cells comprising one more different cell types, or a cell lysate or other proteinaceous mixture comprising one or more glycan binding proteins (GBP). 
     
     
         3 . The method of  claim 2 , wherein the target material is a population of cells comprising two more different cell types, or a cell lysate or other proteinaceous mixture comprising two or more glycan binding proteins (GBP). 
     
     
         4 . The method of  claim 3 , wherein the target material is a population of cells and the target material element(s) is one or more of the cell types therein. 
     
     
         5 . The method of  claim 4 , wherein the one or more cell types preferentially binds to the multivalent glycan scaffold compared to other cell type(s) in the population. 
     
     
         6 . The method of any one of  claims 2-5 , further comprising isolating the multivalent scaffold with the cells bound thereto. 
     
     
         7 . The method of any one of  claims 2-6  further comprising characterizing the bound cells. 
     
     
         8 . A composition comprising the glycan scaffold-bound cells of any one of  claims 2-7 . 
     
     
         9 . The method of  claim 2 , wherein the target material is a cell lysate or other proteinaceous mixture and the target material element is one or more of the GBPs therein. 
     
     
         10 . The method of  claim 9 , wherein the one or more GBPs preferentially binds to the multivalent glycan scaffold compared to other GBPs in the cell lysate or other proteinaceous mixture. 
     
     
         11 . The method of any one of  claims 9 or 10  further comprising characterizing the bound GBP. 
     
     
         12 . The method of any one of  claims 9-10  further comprising isolating the bound GBP. 
     
     
         13 . A composition comprising the glycan scaffold-bound GBP of any one of  claims 9-12 . 
     
     
         14 . A method of identifying glycan-based binding interactions comprising contacting one or more known target material elements with one or more species of multivalent glycan scaffolds each comprising two or more glycans, and identifying glycan scaffold(s) that bind thereto. 
     
     
         15 . The method of  claim 14  comprising contacting the one or more known target material elements with a library of different species of multivalent glycan scaffolds each comprising two or more glycans and identifying glycan scaffold(s) that bind thereto, optionally wherein the library comprises between about 2 to about 10,000 different species of glycan scaffolds, or any subrange or specific integer thereof, such as 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000. 
     
     
         16 . The method of  claim 14 or 15 , wherein the known target material element is one or more cell types. 
     
     
         17 . The method of  claim 16 , wherein one or more of the multivalent glycan scaffolds preferentially bind to the one or more cell types compared to other multivalent glycan scaffolds in the library. 
     
     
         18 . The method of any one of  claims 16 or 17  further comprising characterizing the bound multivalent glycan scaffold(s). 
     
     
         19 . The method of any one of  claims 16-18  further comprising isolating the multivalent glycan scaffold(s). 
     
     
         20 . A composition comprising the glycan scaffold-bound cells of any one of  claims 14-19 . 
     
     
         21 . The method of  claims 14 or 15 , wherein the known target material element is one or more GBP. 
     
     
         22 . The method of  claim 21 , wherein one or more of the multivalent glycan scaffolds preferentially bind to the one or more GBP compared to other multivalent glycan scaffolds in the library. 
     
     
         23 . The method of any one of  claims 21 or 22  further comprising characterizing the bound multivalent glycan scaffold(s). 
     
     
         24 . The method of any one of  claims 21-23  further comprising isolating the multivalent glycan scaffold(s). 
     
     
         25 . A composition comprising the glycan scaffold-bound GBP of any one of  claims 21-24 . 
     
     
         26 . A method of identifying glycan-based binding interactions comprising contacting a target material with one or more unknown multivalent glycan scaffolds comprising two or more glycans, and identifying element(s) from the target material that binds thereto, optionally wherein the one or more unknown multivalent glycan scaffolds form a library comprising between about 2 about 10,000 different species of glycan scaffolds, or any subrange or specific integer thereof, such as 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000. 
     
     
         27 . The method of  claim 26 , wherein the target material is a population of cells comprising one more different cell types, or a cell lysate or other proteinaceous mixture comprising one or more glycan binding proteins (GBP). 
     
     
         28 . The method of  claim 27 , wherein the target material is a population of cells comprising two more different cell types, or a cell lysate or other proteinaceous mixture comprising two or more glycan binding proteins (GBP). 
     
     
         29 . The method of  claim 28 , wherein the target material is a population of cells and the target material element(s) is one or more of the cell types therein. 
     
     
         30 . The method of  claim 29 , wherein the one or more cell types preferentially binds to the multivalent glycan scaffold(s) compared to other cell type(s) in the population. 
     
     
         31 . The method of any one of  claims 26-30 , further comprising isolating the multivalent scaffold(s) with the cells bound thereto. 
     
     
         32 . The method of any one of  claims 26-31  further comprising characterizing the bound cells and/or the glycan(s) bound thereto. 
     
     
         33 . A composition comprising the glycan scaffold-bound cells of any one of  claims 27-32 . 
     
     
         34 . The method of  claim 27 , wherein the target material is a cell lysate or other proteinaceous mixture and the target material element is one or more of the GBPs therein. 
     
     
         35 . The method of  claim 34 , wherein the one or more GBPs preferentially binds to the multivalent glycan scaffold compared to other GBPs in the cell lysate or other proteinaceous mixture. 
     
     
         36 . The method of any one of  claims 34 or 35  further comprising characterizing the bound GBP and/or the glycan(s) bound thereto. 
     
     
         37 . The method of any one of  claims 34-36  further comprising isolating the bound GBP. 
     
     
         38 . A composition comprising the glycan scaffold-bound GBP of any one of  claims 34-37 . 
     
     
         39 . The method or composition of any one of  claims 1-38 , wherein the multivalent glycan scaffold comprises an underlying branched scaffold with two or more glycans and optionally one or more functional elements linked to the terminal ends thereof. 
     
     
         40 . The method or composition of  claim 39 , wherein the underlying branched scaffold is a dendrimer. 
     
     
         41 . The method or composition of  claims 39 or 40 , wherein the two or more glycans are all of the same species of glycan. 
     
     
         42 . The method or composition of  claims 39 or 40 , wherein the two or more glycans are comprised of two or more different species of glycans. 
     
     
         43 . The method or composition of any one of  claims 39-42 , wherein one or more of the functional element(s) is an agent that facilitates formation, imaging, localization, identification, isolation, and/or characterization of the glycan scaffold, and/or the target material element bound thereto. 
     
     
         44 . The method or composition of any one of  claims 39-43 , wherein at least one of the functional elements is a nucleic acid comprising a barcode that specifically identifies the glycan scaffold. 
     
     
         45 . The method or composition of any one of  claims 39-44 , wherein at least one of the functional elements is a fluorophore. 
     
     
         46 . The method or composition of any one of  claims 39-45 , wherein the glycan is linked to 2-amino-N-(2-aminoethyl)-benzamide (AEAB) which is directly or indirectly linked to the terminal ends of the underling branched scaffold. 
     
     
         47 . The method or composition of any one of  claims 39-45 , wherein at least one of the functional elements is an affinity tag. 
     
     
         48 . The method of composition of any one of  claims 39-47 , wherein the glycan is linked to an amino acid which is directly or indirectly linked to the terminal ends of the underlying branched scaffold. 
     
     
         49 . The method of  claim 48 , wherein the amino acid is no part of a polypeptide chain. 
     
     
         50 . The method of  claim 48 , wherein the amino acid is part of a polypeptide chain. 
     
     
         51 . The method of  claim 50 , wherein the polypeptide chain comprises two or more amino acid acids with glycans linked thereto. 
     
     
         52 . The method of any one of  claims 48-51 , wherein the amino acid is serine or threonine. 
     
     
         53 . The method or composition of any one of  claims 1-52 , wherein the glycan scaffold or target material element is immobilized, optionally to a surface or substrate such as bead, a slide, a plate, or a dish. 
     
     
         54 . The method or composition of any one of  claims 1-52 , wherein neither the glycan scaffold or target material are immobilized. 
     
     
         55 . The method or composition of  claim 54 , wherein the glycan scaffold and target material are in solution. 
     
     
         56 . A method of treating a subject in need thereof, comprising identifying a glycan-based binding interaction according to any of the preceding methods and administering the subject a composition comprising an effective amount of a therapeutic multivalent glycan scaffold comprising the glycan of the identified interaction. 
     
     
         57 . The method of  claim 56 , wherein therapeutic glycan induces or inhibits a cellular activity upon binding to cell or GBP of the interaction. 
     
     
         58 . The method of  claim 56 , wherein therapeutic glycan comprise an active agent functionalized thereto. 
     
     
         59 . The method of  claim 58 , wherein the active agent is a nucleic acid, small molecule, or polypeptide. 
     
     
         60 . The method of  claim 59 , wherein the cell is a cancer cell and the active agent is an anti-cancer drug. 
     
     
         61 . The method of  claim 59 , wherein the cell is an immune cell and the active agent is or encodes an antigen. 
     
     
         62 . A composition comprising the therapeutic glycan of any one of claims  56 - 62 . 
     
     
         63 . A multivalent glycan scaffold comprising an underlying branched scaffold with two or more glycans and one or more functional elements linked to the terminal ends thereof. 
     
     
         64 . The multivalent glycan scaffold of  claim 63 , wherein at least one of the functional element(s) is an agent that facilitates formation, imaging, localization, identification, isolation, and/or characterization of the glycan scaffold, and/or a target material element bound thereto. 
     
     
         65 . The multivalent glycan scaffold of any one of  claims 63 or 64 , wherein at least one of the functional elements is a fluorophore. 
     
     
         66 . The multivalent glycan scaffold of any one of  claims 63-65 , wherein the glycan is linked to 2-amino-N-(2-aminoethyl)-benzamide (AEAB) which is directly or indirectly linked to the terminal ends of the underling branched scaffold. 
     
     
         67 . The multivalent glycan scaffold of any one of  claims 63-66 , wherein at least one of the functional elements is a nucleic acid comprising a barcode that specifically identifies the glycan scaffold. 
     
     
         68 . The multivalent glycan scaffold of any one of  claims 63-67 , wherein at least one of the functional elements is an affinity tag. 
     
     
         69 . The method of composition of any one of  claims 63-68 , wherein the glycan is linked to an amino acid which is directly or indirectly linked to the terminal ends of the underlying branched scaffold. 
     
     
         70 . The method of  claim 69 , wherein the amino acid is no part of a polypeptide chain. 
     
     
         71 . The method of  claim 69 , wherein the amino acid is part of a polypeptide chain. 
     
     
         72 . The method of  claim 71 , wherein the polypeptide chain comprises two or more amino acid acids with glycans linked thereto. 
     
     
         73 . The method of any one of  claims 69-72 , wherein the amino acid is serine or threonine. 
     
     
         74 . The multivalent glycan scaffold of any one of  claims 63-73 , wherein the underlying branched scaffold is a dendrimer. 
     
     
         75 . The multivalent glycan scaffold of any one of  claim 63-74 , wherein the underlying scaffold is formed of 2-16 arms, inclusive, optionally 8-16 arms. 
     
     
         76 . The multivalent glycan scaffold of any one of  claim 63-75 , wherein the underlying scaffold and/or arms thereof is formed of polyethylene glycol (PEG) polymer. 
     
     
         77 . The multivalent glycan scaffold of any one of  claim 63-76  wherein the underlying scaffold is formed of 8-Arm PEG-Biotin/Alkyne, Biotin(1)/Alkyne(7), MW 20 k; Dendro Alkyne-PEG-Dendro Alkyne, PEG 10 k, G4 32 Alkyne; Dendro Alkyne-PEG-Dendro Alkyne, PEG 10 k, G5 64 Alkyne; or Dendro Alkyne-PEG-Dendro Alkyne, PEG 10 k, G6 128 Alkyne. 
     
     
         78 . The multivalent glycan scaffold of any one of  claims 63-77 , wherein the multimeric glycan scaffold is between 10 kDa and 50kD, or between 20 kDa and 60 kDa, or between 20 kDa and 30 kDa, each inclusive, or any integer range thereof, or any specific integer therebetween. 
     
     
         79 . The multivalent glycan scaffold of any one of  claims 63-78 , wherein the two or more glycans are all of the same species of glycan. 
     
     
         80 . The multivalent glycan scaffold of any one of  claims 63-79 , wherein the two or more glycans are comprised of two or more different species of glycans. 
     
     
         81 . The multivalent glycan scaffold of any one of  claims 63-80  comprising the formula:
   (Ligand) n :Scaffold:Detection/Separation Tag:Identifier Tag 
 wherein “Ligand” is present and is one or more species of glycan having n >1 copies (i.e., multivalent); 
 “Scaffold” is present and is the underlying branched scaffold, e.g., dendrimer or hyperbranched polymer; 
 one or both of “Detection/Separation Tag” and “Identifier Tag” are present and refer to the functional element e.g., (a) biotin or (HIS)6-8 peptide tag to facilitate separation/isolation, (b) a fluorophore to facilitate detection, (c) a unique DNA or RNA sequence (“barcode”) that can be PCR amplified and sequenced to facilitate identification; and/or (d) a drug or other material(s) as discussed elsewhere herein, e.g., nucleic acids such as DNA, RNA, iRNA, crispr guide RNA, etc., or vesicle containing a drug or such other materials that are intended to have a therapeutic or cell modifying effect or can include an antigen that is intended to induce (a) a protective immune response directed to a pathogen or an aberrant cell, e.g., tumor, that expresses the antigen or (b) immune tolerance to the antigen as a therapy for an autoimmune disorder directed against the antigen. 
 
     
     
         82 . The multivalent glycan scaffold of any one of  claims 63-81 , wherein the glycan scaffold is not immobilized. 
     
     
         83 . A composition comprising a plurality of multivalent glycan scaffold of any of  claims 63-82  in solution. 
     
     
         84 . The composition of  claim 83 , wherein the plurality of multivalent glycan scaffolds are not immobilized.